Molecular cloning of tropomyosins identified as allergens in six species of crustaceans

Although tropomyosin is known to be a major allergen of crustaceans, its structural information is limited to only five species. In this study, tropomyosin was confirmed to be a major allergen in six species of crustaceans (black tiger prawn, kuruma prawn, pink shrimp, king crab, snow crab, and horsehair crab) by immunoblotting. Then, the amino acid sequences of tropomyosins from these crustaceans were elucidated by a cDNA cloning technique. Sequence data for crustacean tropomyosins including the obtained results reveal that fast tropomyosins are contained in shrimps (or prawns) and lobsters, slow tropomyosins in crabs, and both tropomyosins in crayfishes and hermit crabs. Although fast and slow tropomyosins share a high sequence identity (about 90%) with each other, significant differences are observed in specific regions between both tropomyosins.     

Comprehensive analytical method for the determination of hydrophilic metabolites by high-performance liquid chromatography and mass spectrometry

A method for the comprehensive analysis of hydrophilic metabolites, based on a combination of high-performance liquid chromatography and mass spectrometry, is described. We evaluated three types of stationary phases to achieve the separation of highly hydrophilic metabolites. Good chromatographic retention and separation of these metabolites were achieved on a pentafluorophenylpropyl-bonded silica column with gradient elution, using 0.1% aqueous formic acid and acetonitrile as the mobile phase. The optimized conditions allowed the comprehensive determination of the standard 49 kinds of amino acids, 6 kinds of amines, 45 kinds of organic acids, 18 kinds of nucleic bases, 5 kinds of nucleosides, and 14 kinds of nucleotides, and then the linearity, dynamic range, detection limit, and precision of the retention time and the peak area were validated. We applied this method for the targeted analysis of the components in soy sauce. The results from the quantitative determination of amino acids were compared to those obtained with an amino acid analyzer, and the accuracy was in the range between 85 and 119%. The accuracy of other detected components was confirmed to be 105-133% by the recovery rate after the addition of standard compounds. We also applied the method for the nontargeted metabolic profiling of the components in several kinds of soy sauces with the principal component analysis. They were classified by the manufacturing methods, and the components that corresponded to the differences were identified. This method could be useful for the targeted analysis of hydrophilic metabolites as well as their nontargeted metabolic profiling.     

Radical scavenging and singlet oxygen quenching activity of marine carotenoid fucoxanthin and its metabolites

Antioxidant activity of carotenoids is suggested to be one of the factors for their disease preventing effects. Marine carotenoids fucoxanthin and its two metabolites, fucoxanthinol and halocynthiaxanthin, have been shown to exhibit several biological effects. The antioxidant activities of these three carotenoids were assessed in vitro with respect to radical scavenging and singlet oxygen quenching abilities. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity of fucoxanthin and fucoxanthinol was higher than that of halocynthiaxanthin, with the effective concentration for 50% scavenging (EC 50) being 164.60, 153.78, and 826.39 microM, respectively. 2,2'-Azinobis-3-ethylbenzo thizoline-6-sulphonate radical scavenging activity of fucoxanthinol (EC 50, 2.49 microM) was stronger than that of fucoxanthin (EC 50, 8.94 microM). Hydroxyl radical scavenging activity as measured by the chemiluminescence technique showed that the scavenging activity of fucoxanthin was 7.9 times higher than that by fucoxanthinol, 16.3 times higher than that by halocynthiaxanthin, and 13.5 times higher than that by alpha-tocopherol. A similar trend was observed when the hydroxyl radical scavenging was assessed by the electron spin resonance (ESR) technique. ESR analysis of the superoxide radical scavenging activity also showed the superiority of fucoxanthin over the other two carotenoids tested. Singlet oxygen quenching ability of the three carotenoids was lower than that of beta-carotene, with quenching rate constants ( k Q, x10 (10) M (-1) s (-1)) being 1.19, 1.81, 0.80, and 12.78 for fucoxanthin, fucoxanthinol, halocynthiaxanthin, and beta-carotene, respectively. The higher radical scavenging activity of fucoxanthin and fucoxanthinol compared with halocynthiaxanthin is assumed to be due to presence of the allenic bond.     

Juvenile bird acanthocephalans recovered incidentally from raccoon dogs (Nyctereutes procyonoides viverrinus) on Yakushima Island, Japan

Helminth parasites were collected from 14 raccoon dogs (Nyctereutes procyonoides viverrinus) and 2 Japanese weasels (Mustela itatsi sho) on Yakushima Island, Kagoshima Prefecture (Kyushu), Japan. In addition to trematodes and nematodes reported separately, everted cystacanths of 3 bird acanthocephalans (Mediorhynchus robustus in 5 animals, Porrorchis leibyi in 2 animals, and Sphaerirostris lanceoides in 1 animal) were collected from the raccoon dogs introduced recently on this World Natural Heritage Area. A cystacanth of P. leibyi was collected also from a weasel indigenous to this island that were killed by traffic accidents. Recovery of everted cystacanths of M. robustus from the intestine of raccoon dogs was remarkably frequent (36%), suggesting that this acanthocephalan should be highly prevalent in passerine hosts on Yakushima Island, and the animals might vigorously take insect hosts or paratenic hosts. Furthermore, the present report is the second on M. robustus after its first record in Japan (Aichi Prefecture, Honshu) as M. garruli from a Eurasian jay (Garrulus glandarius japonicus) by Yamaguti in 1939. Porrorchis leibyi has never been recorded in Japan.     

Characterization of the reproductive cycle in female bharals (Pseudois nayaur) based on the changes in serum progesterone concentrations and parturition season

The purpose of present study was to determine annual changes in serum progesterone (P(4)) concentrations and to clarify basic reproductive characteristics, such as breeding season, estrous cycle, and puberty in female bharals (Pseudois nayaur). Blood was collected from 9 female bharals once or twice weekly for approximately one year. Serum P(4) concentrations were determined by radioimmunoassay. Serum P(4) concentrations showed remarkable and cyclic changes between November/December (winter) and May/June (late spring). The mean estrous cycle was 24.9 +/- 0.5 days. Chasing insistently to other females and discharge of mucus from the vulva were observed around the time when the serum P(4) concentrations began to increase. The chasing behavior and discharge of mucus were considered to be external indicators of estrus in female bharals. Serum P(4) concentrations of a pregnant female had non-cyclic changes, and the values remained high. In this study, all 37 deliveries were between April and September, and about 70% of these were concentrated in May and June. The conception month determined on the day of birth was between October and April for all animals, and the most common month was in December (54%). This month corresponded to an early stage of the period when the serum P(4) concentrations changed cyclically. These results indicate that many female bharals become pregnant at the beginning of the breeding seasons and, if they do not become pregnant, the estrous cycle, about 25 days in length, is repeated.     

Persorption of luminal antigenic molecule and its specific antibody via apoptotic epithelial cells of intestinal villi and Peyer's patches into peripheral blood in rats

The possibility of persorption of bovine serum albumin (BSA) molecules from mucous epithelial cells and its mechanism were investigated in rats orally pre-immunized by BSA for 14 consecutive days. In the small and large intestines, both the BSA antigen (BSA-Ag) and its specific antibody (SpAb) were absorbed by the epithelial cells at the late apoptotic stage (ApoEp), and were subsequently transcytosed by membranes of the small vesicles. The basal cytoplasms containing highly-concentrated BSA-Ag and SpAb were occasionally fragmented into small cytoplasmic droplets that were secreted into the lamina propria. In Peyer's patches, both BSA-Ag and SpAb were more actively absorbed and transcytosed toward the dome area by the ApoEp of the dome apex than by the M cells. BSA-Ag and SpAb were finally persorbed into the portal blood and lymph, but were never secreted into the bile. They were also engulfed by macrophage-like cells in the villous lamina propria, mesenteric lymph node and spleen, and by hepatocytes in the liver. These findings suggest that sensitized soluble luminal antigens are taken up by ApoEp in the small intestine and are finally persorbed into the peripheral blood. The uptake of luminal antigen might be mediated by its luminal SpAb.     

Development of DNA markers that discriminate between white- and blue-flowers in Japanese gentian plants

We developed molecular markers for discrimination of white and blue flower color in Japanese gentian plants. White-flowered gentians can be classified into two types, based on genetic and physiological features. One type includes four allelic variations (gtmyb3-1, gtmyb3-2, gtmyb3-3, and gtmyb3-4) of an anthocyanin biosynthetic regulator gene (GtMYB3), distinguished by three PCR-based molecular markers. The other type contains a newly identified inactive allele (ans1) of the anthocyanidin synthase (ANS) gene with a premature stop codon generated from a 4-bp deletion in the second exon. The ans1 allele was distinguished from the active ANS allele by a cleaved amplified polymorphism sequence (CAPS) marker. The genotypes of 12 white-flowered gentian cultivars/lines could be identified and classified as either ans1 or gtmyb3 using these four molecular markers. No white-flowered gentians contained ans1 and gtmyb3 alleles simultaneously. The mutated ANS gene co-segregated with white flower color in an F₂ population, demonstrating that the CAPS marker is useful to discriminate between white and blue flowers in gentian. Markers to discriminate flower color in Japanese gentian will be useful for early selection of progeny and for breeding management.     

Cystic cholangioma in the thoracic cavity of a rat

A female congenic rat produced by repeated backcrossing of Nihon rats, a model for hereditary renal cell carcinoma, to Brown Norway rats was necropsied at 24 months of age. At necropsy, a white mass about 1 centimeter in size was observed in the thoracic cavity, and the mass partly adhered to the esophagus and the diaphragm. Histologically, the mass was clearly circumscribed by connective tissue, and consisted of neoplastic cuboidal epithelial cells that showed cystic tubular proliferation. Some islands of well-differentiated hepatocytes and some vessels were observed in the mass. Immunohistochemically, the tumor cells were strongly positive for cytokeratin and partly positive for vimentin but were negative for mesothelin and Von Willebrand Factor. The positive rate for Ki-67 was 2.4%. Based on these histological and immunohistochemical evidences, we diagnosed this tumor as a cystic cholangioma that might have arisen from the ectopic hepatic tissue in the thoracic cavity.     

Changes in circulating adiponectin and metabolic hormone concentrations during periparturient and lactation periods in Holstein dairy cows

Although our previous report demonstrated that adiponectin and AdipoR1 gene expressions changed among different lactation stages in the bovine mammary gland, its in vivo kinetics remain unclear in ruminant animals. In this study, we investigated the changes in circulating concentrations of adiponectin, as well as other metabolic hormones and metabolites, (i) during the periparturient period and (ii) among different lactation stages, in Holstein dairy cows. In experiment 1, serum adiponectin concentrations increased after parturition. Serum insulin concentrations were lower in the postpartum than prepartum period, whereas serum growth hormone (GH) concentrations increased in the postpartum period. Serum nonesterified fatty acids (NEFA) levels were increased during the postpartum period and were dependent on the parity. In experiment 2, there was no significant difference in plasma adiponectin concentrations among lactational stages. Plasma insulin concentrations tended to be lower in early lactation while plasma GH levels tended to be higher. Plasma NEFA concentrations were significantly lower in mid‐ and late‐lactation stages than non‐lactation stages. These findings indicate that elevation of serum adiponectin might be involved in energy metabolism just around parturition, and might exert its action through regulation of receptor expression levels in target tissues in each lactational stage in Holstein dairy cows.